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Autoinhibition of human dicer by its internal helicase domain

Academic Article
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Overview

authors

  • Ma, E.
  • MacRae, Ian
  • Kirsch, J. F.
  • Doudna, J. A.

publication date

  • June 2008

journal

  • Journal of Molecular Biology  Journal

abstract

  • Dicer, a member of the ribonuclease III family of enzymes, processes double-stranded RNA substrates into approximately 21- to 27-nt products that trigger sequence-directed gene silencing by RNA interference. Although the mechanism of RNA recognition and length-specific cleavage by Dicer has been established, the way in which dicing activity is regulated is unclear. Here, we show that the N-terminal domain of human Dicer, which is homologous to DExD/H-box helicases, substantially attenuates the rate of substrate cleavage. Deletion or mutation of this domain activates human Dicer in both single- and multiple-turnover assays. The catalytic efficiency (k(cat)/K(m)) of the deletion construct is increased by 65-fold over that exhibited by the intact enzyme. Kinetic analysis shows that this activation is almost entirely due to an enhancement in k(cat). Modest stimulation of catalysis by the full-length Dicer enzyme was observed in the presence of the TAR-RNA binding protein, which physically interacts with the DExD/H-box domain. These results suggest that the DExD/H-box domain likely disrupts the functionality of the Dicer active site until a structural rearrangement occurs, perhaps upon assembly with its molecular partners.

subject areas

  • Catalysis
  • DEAD-box RNA Helicases
  • Endoribonucleases
  • Humans
  • Kinetics
  • Protein Binding
  • Protein Structure, Tertiary
  • RNA, Double-Stranded
  • RNA-Binding Proteins
  • Ribonuclease III
  • Sequence Deletion
  • Substrate Specificity
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Research

keywords

  • RNAi
  • dicer
  • helicase
  • ribonuclease
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Identity

PubMed Central ID

  • PMC2927216

International Standard Serial Number (ISSN)

  • 0022-2836

Digital Object Identifier (DOI)

  • 10.1016/j.jmb.2008.05.005

PubMed ID

  • 18508075
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Additional Document Info

start page

  • 237

end page

  • 243

volume

  • 380

issue

  • 1

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